Vaccine against gram negative bacteria

ABSTRACT

A mucosal vaccine for Neisseria based on native outer membrane vesicles (NOMV) prepared from genetically engineered vaccine strain and unexposed to detergents is described. Methods for extending this vaccine approach to other Gram negative bacteria are also decribed.

INTRODUCTION

[0001] Group B meningococcal disease currently accounts for at least onehalf of all meningococcal disease in many countries including North andSouth America, and Europe. The emergence of a new virulent clone ofgroup B Neisseria meningitidis, known as ET5, in Norway in the late 70'shas since been responsible for prolonged epidemics in Norway, Cuba,Brazil, and Chile. These epidemics have created serious public healthproblems and led to intensive efforts to develop an effective group Bvaccine in several of the affected countries. Recently, an outbreak ofgroup B disease caused by the ET5 clone occured in the U.S. Northwest.This could indicate that the ET5 clone is gaining a foothold in the U.S.and that an increase in the incidence of group B disease in the U.S. mayoccur in the next few years. The absence of a U.S.-licensed group Bvaccine along with the poor performance of the A and C capsularpolysaccharide vaccines in children under 18 months have preventedserious consideration of routine childhood vaccination againstmeningococcal disease. Current efforts to develop conjugate group A andC polysaccharide vaccines by several companies will likely yield A and Cvaccines with improved performance in young children and will makeroutine vaccination of children against meningococcal disease moreattractive, particularly if an effective group B vaccine becomesavailable.

[0002] Neisseria, including Neisseria meningitidis and Neisseriagonorrhoeae have an outer membrane that is rather loosely associatedwith the rigid cell wall peptidoglycan layer and naturally blebs offduring growth of the organism [Zollinger, W. D. et al. (1972) Infect.Immun. 6:835-851] (All documents cited herein supra and infra are herebyincorporated by reference thereto.). Vesicles of outer membrane can beobtained from a meningococcal culture supernatant or by extraction fromthe organism by mild procedures [Zollinger, W. D., ibid.]. Thesevesicles appear to be representative of intact outer membrane and can beeasily obtained in high yield. Although an excellent antigen, the use ofthese vesicles directly as a vaccine against meningococcal disease hasbeen considered impractical because of the high levels oflipopolysaccharide (a potent endotoxin) associated with them. Typically,the weight ratio of LPS to protein in the outer membrane is about 0.3 to0.8. For many years, efforts have been made to utilize the outermembrane proteins as a vaccine for group B meningococcal disease byusing detergents to remove most of the lipopolysaccharide from the outermembrane. These candidate vaccines have been partially successful (50 to80% efficacy in field trials), but have failed to induce protectiveantibody responses in children under the age of four years. Protectioninduced by these vaccines also seems to be of limited duration. Theyoung children have IgG antibody responses against the outer membraneproteins that equals or exceeds those of older children, but most of theantibodies are not bactericidal or protective.

[0003] Therefore, there is a need for a Neisseria meningitidis vaccinewhich produces a lasting, protective immunogenic response capable ofprotecting an individual against meningococcal disease.

SUMMARY

[0004] The present invention fulfills the need described above.

[0005] The present invention relates to a vaccine and methods ofproducing a vaccine or vaccines that can be used to immunize anindividual against meningococcal disease, and in the extendedapplication, against other Gram negative infections with bacteria suchas Shigella, Brucella, Pseudomonas, E. coli, and Haemophilus.

[0006] The vaccine of the present invention introduce the outer membraneproteins (OMPs) in their natural phospholipid/lipopolysaccharide (LPS)environment as native outer membrane vesicles (NOMV), and results in animproved functional (bactericidal) antibody response to the outermembrane proteins (OMPs) in animals and should behave similarly inhumans and children under the age of 4 years. We have shown that thistype of vaccine, which is normally considered to be too toxic for use asa parenteral vaccine, can be safely administered via the intranasalroute. Intranasal immunization is ideal for meningococcal vaccines sinceasymptomatic nasopharyngeal colonization by less pathogenic meningococciand closely related species results in natural immunization of mostindividuals. The human nasopharynx is the natural habitat of N.meningitidis and at any given time approximately 5 to 10% of healthyindividuals carry it on their throats.

[0007] There are several advantages to using a native outer membranevesicle as an intranasal vaccine for N. meningitidis or other Gramnegative infection. NOMV can be prepared easily at relatively low costso the technology and the product produced by it may be more accessibleto under-developed countries.

[0008] The antigens presented as part of NOMV, including the OMPs andthe LPS, are in a completely native configuration and environment aspart of intact outer membrane that has not been exposed to detergent.This results in an antibody response that is directed primarily towardepitopes exposed on the surface of the intact bacterium. Theseantibodies are more likely to be functional than if directed againstepitopes that are conformationally altered (by detergent extraction forexample), or not fully exposed at the surface of the viable organism. Inaddition, giving the NOMV vaccine intranasally results in very littlereactogenicity or toxicity in spite of relatively high endotoxincontent.

[0009] The intranasal route of vaccination mimics the natural route ofimmunization for N. meningitidis and, as judged by the results of animalexperiments, is expected to induce a mucosal immune response as well asa serum antibody response. Antibodies to antigens such as Opc and Opaproteins and pili may play a more important role in protection at themucosal surface during the initial phases of pathogenesis than they doin the serum where the organism may have turned off their expression.The nasopharynx is the natural portal of infection for themeningococcus.

[0010] In the youngest children who have had little exposure tomeningococci, it may be particularly important to prime the immunesystem with a very native antigen that can induce protective antibodiesso that subsequent colonization with meningococci will be effective inboosting protective immunity. In contrast, isolated OMPs given as avaccine tend to induce a preponderance of antibody that is nonfunctionalin a bactericidal assay.

[0011] In particular, the present invention relates to the use of NOMVfrom a vaccine strain that has been genetically modified in order toproduce maximum immunogenicity with least toxicity. More specifically, avaccine strain of the present invention includes a modified strain whichdoes not synthesize sialic acid (capsule and sialylated LPS) resultingin better interaction of the NOMV with the mucosal surface and producinghigher immunogenicity.

[0012] Preferably, the vaccine strain is grown under iron limitingconditions to induce the expression of the iron uptake proteinsincluding transferrin and lactoferrin binding proteins resulting in avaccine that contains additional antigens with known protectivepotential both by inducing bactericidal antibodies and by inducingantibodies that can block the binding of iron by the organism.

[0013] Therefore, it is an object of the present invention to provide aNeisseria vaccine comprising purified native outer membrane vesicles ofNeisseria in an amount effective to elicit protective antibodies in ananimal to Neisseria; and a pharmaceutically acceptable diluent, carrier,or excipient.

[0014] It is another object of the present invention to provide aNeisseria meningitidis vaccine comprising purified native outer membranevesicles of Neisseria meningitidis in an amount effective to elicitprotective antibodies in an animal to Neisseria meningitidis; and apharmaceutically acceptable diluent, carrier, or excipient

[0015] It is yet another object of the present invention to provide aNeisseria meningitidis Group B vaccine comprising purified native outermembrane vesicles of Neisseria meningitidis Group B in an amounteffective to elicit protective antibodies in an animal to Neisseriameningitidis; and a pharmaceutically acceptable diluent, carrier, orexcipient

[0016] It is another object of the present invention to provide a methodof preparing a Neisseria vaccine comprising isolating native outermembrane vesicles from Neisseria.

[0017] It is yet another object of the present invention to provide amodified strain of N. meningitidis for use in the production of nativeouter membrane vesicles wherein the modified strain is unable tosynthesize sialic acid.

[0018] It is further another object of the present invention to providea modified strain of N. meningitidis for use in the production of nativeouter membrane vesicles wherein the modified strain is unable tosynthesize sialic acid and expresses iron uptake proteins.

[0019] It is yet a further object of the present invention to provide amethod for the production of N. meningitidis expressing iron uptakeproteins by growing N. meningitidis in iron deficient media.

[0020] It is a further object of the present invention to provide astrain of N. meningitidis 9162 synX(−).

[0021] It is another object of the present invention to provide a methodfor the preparation of native outer membrane vesicles comprisingextraction of outer membrane vesicles from cells without exposure todetergent, followed by differential centrifugation, treatment of extractsupernatant with ion exchange matrix, and ultrafiltration.

[0022] It is still another object of the present invention to provide amethod for the preparation of outer membrane vesicles from Gram negativebacteria other than Neisseria for use as a vaccine, the methodcomprising deleting lpp and ompA genes or their equivalents in thebacteria.

[0023] It is yet another object of the present invention to provide avaccine comprising native outer membrane vesicles of Gram negativebacteria other than Neisseria, produced according to the above methods,in an amount effective to elicit protective antibodies in an animal tothe Gram negative bacteria and a pharmaceutically acceptable diluent,carrier, or excipient.

[0024] Further objects and advantages of the present invention will beclear from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1. Bactericidal antibody response of rabbits vaccinatedintranasally with meningococcal NOMV vaccines. Groups of 4 rabbits werevaccinated intranasally at 0, 4 and 8 weeks with either 9162 NOMV fromthe encapsulated parent strain, lot # D190894, (group 1) or 9162 synX(−)NOMV, Lot # E110195, (group 2). A control group received only normalsaline. Rabbits were bled and nasal washes taken at 0, 14, 28, 42, 56,and 70 days. The encapsulated parent strain 9162 was used as the teststrain in the bactericidal test. Values are geometric mean reciprocaltiters and the error bars represent 1 standard error of the geometricmean.

[0026]FIG. 2. IgA antibody to NOMV measured by enzyme linkedimmunosorbant assay (ELISA) in the nasal washes of rabbits vaccinatedintranasally with NOMV vaccines. Rabbits were vaccinated as indicated inthe description of FIG. 1. NOMV from the synX(−) mutant strain was usedas the antigen. Values are geometric mean reciprocal values expressed asELISA units (the mean of the product of the dilution and the opticaldensity at 405 nm taken at 2 or 3 points in the optical density range of0.1 to 1.0). The error bars represent 1 standard error of the geometricmean.

[0027]FIG. 3. Antibody to meningococcal lipooligosaccharide (LOS) insera of rabbits vaccinated intranasally with NOMV vaccines. Rabbits werevaccinated as indicated in the description of FIG. 1. Purified LOS ofimmunotypes L3,7 and L8 were used as antigen in the assay. Results areexpressed as geometric mean micrograms per milliliter of specificantibody. The error bars represent 1 standard error of the geometricmean.

[0028]FIG. 4. Electron micrograph of NOMV lot #0123 taken after negativestaining with phosphotungstic acid. Magnification about 77,000 ×.

[0029]FIG. 5. Polyacrylamide gel electrophoresis of NOMV lot #0123 andprotein molecular weight standards. The asterisks indicate the proteinsthat were induced by iron starvation. The arrows at the right identifyprincipal outer membrane protein antigens: Tbp2=transferrin bindingprotein 2, PorA=porin A also called the class 1 protein or subtypeprotein, PorB=porin B also called the class 3 or serotype protein,Rmp=reduction modifiable protein or class 4 protein, Opa=opacityproteins (2 are indicated) or class 5 protein.

[0030]FIG. 6. Bactericidal antibody response of mice to intranasalvaccination with 9162 synX(−) NOMV lot #0123. Groups of 5 mice werevaccinated intranasally on days 0 and 28 with 25 μl of vaccinecontaining the indicated amount of NOMV. A separate group was vaccinatedwith normal saline as a control. Pre-vaccination levels measured on aseparate group were <1:2. The mice were bled on days 28 and 42. Themouse sera were tested against the encapsulated parent strain 9162 usingnormal human serum as a source of complement.

[0031]FIG. 7. Bactericidal antibody response of mice to intraperitonealvaccination with 9162 synX(−) NOMV lot #0123. Groups of 5 mice werevaccinated intraperitoneally on days 0 and 28 with 100 μl of vaccinecontaining the indicated amount of NOMV. A separate group was vaccinatedwith normal saline as a control. Pre-vaccination levels measured on aseparate group were <1:2. The mice were bled on days 28 and 42. Themouse sera were tested against the encapsulated parent strain 9162 usingnormal human serum as a source of complement.

[0032]FIG. 8. Serum IgG antibody response of mice to intranasalvaccination with NOMV Lot #0123 by ELISA. NOMV from the parent 9162strain was used as the antigen. Mice were vaccinated as indicated indescription of FIG. 6. Data are expressed as the geometric mean ELISAunits (see description for FIG. 2) of specific IgG antibody permilliliter of serum. The bars indicate one standard error of the mean.

[0033]FIG. 9. Serum IgG antibody response by ELISA of mice tointraperitoneal vaccination with NOMV Lot #0123. NOMV from the parent9162 strain was used as the antigen. Mice were vaccinated as indicatedin description of FIG. 6. Data are expressed as the geometric mean ELISAunits (see description for FIG. 2) of specific IgG antibody permilliliter of serum. The bars indicate one standard error of the mean.

[0034]FIG. 10. Serum IgA antibody response of mice to intranasalvaccination with NOMV Lot #0123 by ELISA. NOMV from the parent 9162strain was used as the antigen. Mice were vaccinated as indicated indescription of FIG. 6. Data are expressed as the geometric meanmicrograms of specific IgA antibody per milliliter of serum. The barsindicate one standard error of the mean.

[0035]FIG. 11. Serum IgA antibody response by ELISA of mice tointraperitoneal vaccination with NOMV Lot #0123. NOMV from the parent9162 strain was used as the antigen. Mice were vaccinated as indicatedin description of FIG. 6. Data are expressed as the geometric meanmicrograms of specific IgA antibody per milliliter of serum. The barsindicate one standard error of the mean.

[0036]FIG. 12. Polyacrylamide gel electrophoresis of NOMV purified bydifferent methods. The gel was stained with Coomassie Blue. Lanes 1 and5 are molecular weight standards with molecular weights as shown in FIG.5; lane 2, NOMV purified by 3 cycles of ultracentrifugation; lane 3,NOMV purified by new DEAE/Microfiltration method; lane 4. NOMV purifiedby new DEAE/ultrafiltration method.

[0037]FIG. 13. Polyacrylamide gel electrophoresis of NOMV purified fromE. coli lpp(−) ompA(−) with double mutation in outer membrane proteinsand a loose outer membrane phenotype. The gel was stained with CoomassieBlue. Lane 1 is molecular weight standards and lane 2 is E. coli NOMV.

DETAILED DESCRIPTION

[0038] The present invention relates to a vaccine for Neisseria based onnative outer membrane vesicles (NOMV) and to methods for preparing sucha vaccine and extending this vaccine approach to the preparation ofsimilar vaccines against other Gram negative bacteria.

[0039] More particularly, the vaccine for Neisseria meningitidis Group Bdescribed in this invention results in an improved functional(bactericidal) antibody response to the outer membrane proteins (OMPs)in animals and is expected to give an improved bactericidal antibodyresponse in humans, including children under the age of 4 years, bypresenting the OMPs in their natural phospholipid/lipopolysaccharide(LPS) environment as native outer membrane vesicles (NOMV). This type ofvaccine, which is normally considered to be too toxic for use as aparenteral vaccine, can be safely administered via the intrarasal route.Intranasal vaccination against N. meningitidis infections is expected tobe an effective route of vaccination because it mimics the process ofnatural immunization and will induce secretory antibodies at the mucosalsurface that may be able to prevent adhesion and/or invasion at themucosal surface, in addition to inducing bactericidal antibodies in theserum. This type of vaccine will be simple and inexpensive tomanufacture and should therefore be accessible to populations at risk inpoor underdeveloped countries.

[0040] Native outer membrane vesicles can be prepared from any strain ofNesseria, including N. meningitidis, N. gonorrhoeae, and N. lactamica,expressing the most desirable antigens and suppressing expression ofantigens that may interfere with the desired immune response. Desirableantigens would include for example, antigens that have been shown toinduce serum bactericidal antibodies, or antibodies that block uptake ofiron and thereby prevent growth, or antibodies that prevent invasion atthe mucosal surface. Among these are PorA, PorB, Opc, transferrin andlactoferrin binding proteins, LPS and others that are less wellcharacterized. Because antigenic variation occurs with most of theseantigens, it would likely be desirable to cause expression of multipleantigenically different copies of certain proteins such as PorA [Ley P Avan der et al. (1995) Vaccine 13: 401-407] or TbpB.

[0041] Antigens which are not desirable would include the group Bcapsule sialylated LPS antigens which have been shown to inhibitadherence to and invasion of epithelial and or endothelial cells byviable meningococci suggesting that interaction of the NOMV with themucosal cell surface may be decreased by the presence of capsularpolysaccharide and/or sialylated LPS on the NOMV.

[0042] Therefore, it may be advantageous to delete genes coding for thereduction modifiable protein (Rmp) which has been shown to induceantibodies that may block bactericidal activity of other antibodiesunder certain conditions. It appears to be desirable to block sialicacid synthesis and thereby prevent expression of capsule (in N.meningitidis groups B, C, Y and W135) and sialylated LPS. One may alsowant to block expression of LPS types that include theLacto-N-neotetraose group which is crossreactive with precursors ofblood group antigens.

[0043] A further desirable characteristic of the vaccine strain is theexpression of Opc protein which is known to function as an adhesinand/or an invasin. It is likely that this protein is able to bind tospecific receptors on the cells at the mucosal surface and therebyenhance the immunogenicity of the vaccine. This outer membrane proteinis subject to phase variation in expression. Mutants with constitutiveexpression of this protein can be prepared for example, by replacing thermp protein which has stable expression, but may be an undesirablevaccine component, with a copy of the opc gene [van der Ley, P et al.1995, ibid.; van der Ley, P. et al. (1992) Infect. Immun. 60: 3156-3161;Aho et al. (1991) Mol. Microbiol. 5: 1429-1437; Olyhoek et al. (1991)Microbial Pathogenesis 11: 249-257; Sarkari et al. (1994) Mol.Microbiol. 13: 207-217; Klugman et al. (1989) Infect. Immun. 57:2066-2071]

[0044] The usefulness of the Opc protein as antigen in a NOMV intranasalvaccine is greater than in a parenteral vaccine because its expressionmay be necessary for adherence and invasion at the mucosal surface eventhough it may be later turned off when the bacterium enters the bloodstream and becomes subject to killing by serum antibody and complement.It is also clear that NOMV prepared from certain multivalent vaccinestrains developed by other investigators could be used in thepreparation of this type of vaccine. Multivalent strains such as strains1-2B-, PL16215, PL10124, and PL9146 described by van der Ley et al.[1995, ibid.] or the PorA hybrid strains TR516 or TR7216 described byvan der Ley [1993, ibid.] could be used to prepare NOMV vaccines with abroader range of protection. The multivalent PorA strains described inthe first reference or similar strains are already being used to preparevaccines based on deoxycholate extracted vesicles for parenteral use.The present invention would allow the more native NOMV vaccine to bemade from these strains. If an analog of the htrB mutation described inE. coli [Karow et al. (1991) J. Bacteriol. 173: 743-750] and Haemophilisinfluenzae [Lee et al. (1995) J. Biol. Chem. 270: 27151-27159] wereintroduced into the vaccine strains the resultant mutants would beexpected to be defective in lipid A biosynthesis and produce LPS (outermembrane) with much reduced endotoxin activity. The resulting NOMV wouldlikely be safe for parenteral use and would likely be superior to thepresent vaccines made by deoxycholate extraction. A parenteral vaccineis one that is injected either intramuscularly, subcutaneously, orintradermally. A mucosal vaccine is one that is applied to appropriatemucosal surfaces such as the nose and throat, the lung, the gut (oralvaccine), the vagina, or the rectum. A vaccine that is safe as a mucosalvaccine may not be safe for parenteral use.

[0045] Accordingly, we have produced NOMV from a mutant vaccine strainthat cannot synthesize sialic acid and therefore cannot make capsule norsialylate its LPS. NOMV from this mutant strain were found to induce ahigher bactericidal antibody response in rabbits vaccinated intranasallythan NOMV from an isogenic wild type strain.

[0046] We have developed appropriate growth media and culture conditionsthat allow growth of the vaccine strain under conditions of limitingiron availability which results in expression of the iron uptakeproteins including transferrin binding protein 2 and still results in arelatively good yield of bacteria. In addition, the LPS content of theNOMV from meningococci grown under these conditions is only about 20%relative to protein as compared to an expected 30% to 80% thus resultingin lower toxicity.

[0047] The preparation and use of this type of vaccine for other Gramnegative infections is likely to be effective and have a number ofadvantages over current vaccine candidates. For example, NOMV preparedfrom a strain of Neisseria gonorrhoeae that lacks protein III andexpresses the Opa protein has been shown to mediate adherence toepithelial cells [Virji, M. et al. (1993) Mol. Microbiol. 10:499-510]and may be an effective vaccine against gonococcal disease when givenintranasally or in the vagina or rectum [Plummer et al. (1994) J. Clin.Invest. 93: 1748-1755; Waldbeser et al. (1994) Mol. Microbiol. 13:919-928; Kupsch et al. (1993) EMBO J. 12: 641-650]. N. gonorrhoeae NOMVfrom cells grown on solid or liquid medium can be produced usingsubstantially the same procedures as for the meningococcus. Gonococcalvaccines that have been investigated thus far have been based onpurified cell surface components such as pili and the outer membraneprotein PorB. The LPS has also been shown to induce bactericidalantibodies. One study used parenteral priming with a synthetic peptidevaccine followed by boosting with a whole cell oral vaccine [Trees etal. (1994) Proceedings of the Ninth International Pathogenic NeisseriaConference, Winchester, England September 26-30, p. 841]. The use ofNOMV as a vaginal or rectal vaccine would probably require that thevaccine be combined with a gel or thick emulsion so that it would stayin place for a period of time when applied to the mucosa at those sites.

[0048] Extension of this vaccine concept to other Gram negative bacteriaother than Neisseria would be a little more complex but within the skillof a person in the art The main issue for other (non-Neisseria) Gramnegative bacteria is that the outer membrane is much more tightlyassociated with the peptidoglycan layer than is the case for theNeisseria. It is therefore more difficult to prepare substantialquantities of outer membrane vesicles from these organisms. Several ofthe outer membrane proteins that are missing or different in Neisseriaare important in the close association of the outer membrane and thecell wall peptidoglycan of other species. In E. coli, for example, oneof these is the lipoprotein discovered by Braun [Braun, V. (1975)Biochim. Biophys. Acta 415:335-377] that is coded for by the lpp geneand is covalently linked to the peptidoglycan at the carboxy terminus.This protein, which appears to be missing in Neisseria, is substitutedat the amino terminus with lipid that integrates in the phospholipidbilayer of the outer membrane. A second protein in E. coli is OmpA whichconsists of two main parts or domains. One part loops in and out of theouter membrane and the second part connected by a hinge region protrudesdown into the periplasmic space and interacts with the peptidoglycan. Apaper published in 1978 by Sonntag et al. [J. Bacteriol. 136:280-285]described a double mutant lacking both the lpp coded lipoprotein andOmpA This mutant grew reasonably well on media containing increasedlevels of magnesium and exhibited a circular morphology with abundantblebbing of the outer membrane which appeared in electron micrographs tobe very loosely associated with the rigid peptidoglycan layer. Thus, byintroducing mutations into a prospective vaccine strain of E. coli orother Gram negative bacterium that knocks out the Braun lipoprotein andOmpA proteins (or equivalent OMPs in other species) one could easilyobtain good yields of outer membrane vesicles using substantially thesame methods used for Neisseria.

[0049] The LPS of a number of Gram negative pathogens has beenidentified as a dominant antigen capable of inducing protectiveantibodies, but it is typically a poor immunogen by itself. Thus effortshave been made to covalently conjugate the LPS to a carrier protein ornon-covalently complex the LPS to proteosomes in order to enhance itsimmunogenicity. In these cases the use of NOMV prepared from thehomologous pathogen may be a highly effective solution to the problem ofincreasing the LPS immunogenicity.

[0050] This invention also relates to a more efficient method forproducing NOMV. A general method for preparing NOMV from N. meningitidiswas developed and published a number of years ago [Zollinger, W. D etal. (1979) J. Clin. Invest. 63:836-848]. This method has beensuccessfully used for many years, but is not highly conducive to scaledup production because of two required ultracentrifugation steps.

[0051] We have currently demonstrated that the ultracentrifugation stepscan be eliminated by use of batch-wise adsorption of nucleic acid onto aDEAE ion exchange matrix followed by filtration to remove the ionexchange matrix and then utlrafiltration using a membrane having aMWCO>500,000 or microfiltration using a membrane with a pore size of 0.1μm or less, for example, an AG Technology Corp., Needham Mass.,Ultrafiltration cartridge CFP-1-E-6A. The ultracentrifugation steps wereused to separate the NOMV from the smaller or more soluble nucleic acid,capsular polysaccharide (if expresed), periplasmic and other solubleproteins. Attempts to collect the NOMV without first doing the DEAEtreatment were unsuccessful, resulting with NOMV which were not free ofnucleic acid. The use of the correct membrane size is important and ispreferably between the largest ultrafiltration pore size of about500,000 MWCO and the smallest microfiltration pore size of about 0.1 μm.The soluble proteins, residual nucleic acid, and soluble capsularpolysaccharide pass through the membrane while the vesicles which areapproximately 0.1 μm diameter are retained and can be concentrated. Thisimprovement in processing decreases substantially the time and cost ofpreparing large quantities of NOMV.

[0052] In one embodiment, the present invention relates to a vaccine forprotection against N. meningitidis. The vaccine comprises native outermembrane vesicles from N. meningitidis. The vaccine can be prepared byisolating native outer membrane vesicles from the organism or from theculture medium by methods known in the art or methods described in thisapplication. The purified NOMVs are prepared for administration tomammals by methods known in the art, which can include filtering tosterilize the solution, diluting the solution, adding an adjuvant andstabilizing the solution. Adjuvants which enhance production of NOMVspecific antibodies include, but are not limited to, various oilformulations such as stearyl tyrosine (ST, see U.S. Pat. No. 4,258,029),the dipeptide known as MDP, saponin, aluminum hydroxide, and lymphaticcytokine. Mucosal adjuvants include cholera toxin B subunit (CTB), aheat labile enterotoxin (LT) from E. coli (a genetically toxoided mutantLT has been developed), and Emulsomes (Pharmos, LTD., Rehovot, Israel).

[0053] The adjuvant alum (aluminum hydroxide) or ST may be used foradministration to humans.

[0054] The vaccine can be lyophilized to produce a vaccine against N.meningitidis in a dried form for ease in transportation and storage.Further, the vaccine may be prepared in the form of a mixed vaccinewhich contains the NOMVs described above and at least one other antigenas long as the added antigen does not interfere with the effectivenessof the vaccine and the side effects and adverse reactions are notincreased additively or synergistically. The vaccine can be associatedwith chemical moieties which may improve the vaccine's solubility,absorption, biological half life, etc. The moieties may alternativelydecrease the toxicity of the vaccine, eliminate or attenuate anyundesirable side effect of the vaccine, etc. Moieties capable ofmediating such effects are disclosed in Remington's PharmaceuticalSciences (1980). Procedures for coupling such moeities to a molecule arewell known in the art.

[0055] The vaccine may be stored in a sealed vial, ampule or the like.The present vaccine can generally be administered in the form of a sprayfor intranasal administration, or by nose drops, inhalants, swabs ontonsils, or a capsule, liquid, suspension or elixirs for oraladministration. In the case where the vaccine is in a dried form, thevaccine is dissolved or suspended in sterilized distilled water beforeadministration. Any inert carrier is preferably used, such as saline,phosphate buffered saline, or any such carrier in which the NOMV vaccinehas suitable solubility.

[0056] Generally, the vaccine may be administered orally,subcutaneously, intradermally or intramuscularly but preferablyintranasally or orally in a dose effective for the production ofneutralizing antibody and resulting in protection from infection ordisease. The vaccine may be in the form of single dose preparations orin multi-dose flasks which can be used for mass vaccination programs.Reference is made to Remington's Pharmaceutical Sciences, Mack PublisingCo., Easton, Pa., Osol (ed.) (1980); and New Trends and Developments inVaccines, Voller et al. (eds.), University Park Press, Baltimore, Md.(1978), for methods of preparing and using vaccines.

[0057] In another embodiment, the present invention relates to a methodof reducing N. meningitidis infection symptoms in a patient byadministering to said patient an effective amount of NOMV antibodiesincluding those made in humans, either polyclonal or combinations ofmonoclonals to NOMV, as described above. When providing a patient withNOMV antibodies, the dosage administered will vary depending upon suchfactors as the patient's age, weight, height, sex, general medicalcondition, previous medical history, etc. In general, it is desirable toprovide the recipient with a dosage of the above compounds which is inthe range of from about 1 pg/kg to 500 mg/kg (body weight of patient),although a lower or higher dosage may be administered.

[0058] Described below are examples of the present invention which areprovided only for illustrative purposes, and not to limit the scope ofthe present invention. Other suitable modifications and adaptations ofthe variety of conditions and parameters normally encountered in thisart which are obvious to those skilled in the art are within the spiritand scope of the present invention.

[0059] The following methods and materials were used in the examplesbelow.

[0060] Bacterial Strains. The N. meningitidis strain 9162(B:15:P1.3:P5.10,NT:L3,7) is a case isolate from the CSF of a patient inIquique, Chile in 1990. Beginning with this strain a mutant wasconstructed by deleting a portion of the synX gene and inserting akanamycin marker. SynX is a gene in the biosynthetic pathway of sialicacid, and the mutation resulted in the inability to make the group Bcapsule or to sialylate the LOS. This strain 9162 synX(−) was used asthe vaccine strain for most of the examples given.

[0061] The synX(−) mutant was constructed based on results and sequenceinformation reported by Swartley and Stephens [Swartley and Stephens(1994) J. Bacteriol. 176: 1530-1534] who showed that insertion of atransposon, Tn916, into the synX gene led to a capsule negativephenotype. The same or an equivalent mutation could quite easily beintroduced into any transformable N. meningitidis strain by someonetrained in the art. A plasmid for use in transforming meningococci wasconstructed using the following procedure. Three DNA sequences werepieced together using the splicing by overlap extension (SOE) polymerasechain reaction (PCR) technique [Horton et al. (1989) Gene 77: 61-65].The three DNA sequences included, in order beginning at the 5′ end,synXB bases 67 to 681; the kanamycin resistance gene from pUC4K(Pharmacia LKB Biotech Co.) 671 to 1623; and synXB bases 886 to 1589. Inaddition, at the 5′ end, a putative uptake sequence, ACCGTCTGAA, wasadded by including it at the end of the PCR primer used to amplify thesynXB 67 to 691 base sequence. The complete construct was amplified byPCR, purified and blunt ligated into pUC19. pUC19 was used to transformEscherichia coli DH5α and selected on LB agar with 50 μg kanamycin. Akanamycin resistant colony was selected, the DNA extracted, purified,and cut with XbaI. Another copy of the presumptive uptake sequence wasligated into this multiple cloning region site and the resulting plasmidagain used to transform E. coli DH5α and kanamycin resistant coloniesscreened by PCR for presence of the additional uptake sequence. PlasmidDNA was isolated from a selected colony and used as a template for PCRusing primers that amplified only the insert Fart of the plasmidexcluding the ampicillin resistance gene which should not be introducedinto N. meningitidis. The amplified DNA was then purified and used totransform N. meningitidis strain 9162. The synX(−) mutant of N.meningitidis was selected by kanamycin resistance and confirmed by PCRamplification of the modified region.

[0062] The Lpp(−) OmpA(−) double mutant of E. coli was obtained from Dr.Ulf Henning, Tuebingen, Germany and is described in a paper by Sonntag,et al. [Sonntag, I et al. (1978) J. Bact. 136:280-285].

[0063] Growth media. Meningococci were plated from frozen stocks on GCagar medium (Difco Laboratories, Detroit, Mich.) with 1% v/v definedsupplement [Schneider, H. et al. (1988) Infect. Immun. 56:942-946].

[0064] The growth of Neisseria meningitidis in liquid medium withlimiting iron to induce expression of the iron uptake proteins was doneusing medium with the following composition and without the specificaddition of an iron chelator such as desferol. The medium is modifiedfrom that published by B W Catlin [Catlin B W. (1973) J. Infect. Dis.128:178-194] by replacing several individual amino acids with 1%casamino acids (certified, Difco Laboratories). The medium contained perliter, 0.4 g NH₄Cl, 0.168 g KCl, 5.85 g NaCl, 1.065 g Na₂HPO₄, 0.17 gKH₂PO₄, 0.647 g sodium citrate, 6.25 g sodium lactate (60% syrup), 0.037g CaCl₂.2H₂O, 0.0013 g MnSO₄.H₂O, 5 g glycerol, 0.02 g cysteine, 10 gcasamino acids, 0.616 g MgSO₄, and distilled water to one liter. Thesame iron deficient medium was used for the starter flasks and the finalculture flasks or fermenters.

[0065]Escherichia coli cultures were grown on Luria broth or Luria agar(Difco Laboratories).

[0066] Buffers. TES buffer for extraction of NOMV from pelleted cellscontained 0.05 M Tris-HCl, 0.01 M EDTA, 0.15 M NaCl, pH 7.5.

[0067] Serological Assays. Bactericidal assays were performed asdescribed by Moran et al. [Moran, et al. (1994) Infect. Immun.62:5290-5295] using the parent encapsulated strain 9162 as the teststrain. ELISA assays were performed according to the quantitative methoddescribed by zollinger et al. [(1986) In: N. R. Rose and H. Friedman[Eds.] Manual of Clinical Immunology, Third Edition, American SocietyFor Microbiology, Washington, D.C., p. 346]. Where reagents wereavailable to set up a standard curve, the results were calculated as μgantibody per ml. In other instances the results are reported as ELISAunits which was calculated as the product of the optical density and thereciprocal dilution. In both instances the result was calculated for twoor three points in the linear range of the curve (approximate OD range1.0 to 0.15) and the average reported.

[0068] Polyacrylamide gel electrophoresis. The NOMV preparations wereanalyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis bythe method of Laemmli [Laemmli, U.K. (1970) Nature (London);227:680-685].

EXAMPLE 1

[0069] Small Scale Production of NOMV from Strains 9162 and 9162 synX(−).

[0070] A. Growth of N. meningitidis Strains 9162 and 9162 synX(−)

[0071] The bacterial strains were stored as frozen cultures in skimmilk. The same method was used to prepare NOMV from the parent strain9162 and the mutant sialic acid deficient strain 9162 synX(−). Thestrain to be grown was streaked for isolation on agar GCDS plates andincubated overnight (14-16 hrs) at 37° C. in a candle extinction box.The following day, the growth from about half a plate comprising mostlyisolated colonies was used to inoculate 100 ml of sterile modifiedCatlin's medium without iron in a 500 ml Erlenmeyer flask. The flask wasplaced on a rotary shaker at 37° C. and 180 rpm and allowed to grow for4 to 6 hours. At the end of that time the purity and optical density at600 nm of the cultures was verified and the contents of the flask usedto inoculate one liter of sterile modified Catlin's medium without ironin a 2800 ml Fernbach flask. The culture was grown on a rotary shaker at37° C. and 120 to 180 rpm for 8 hours. The culture was checked forpurity by Gram stain, inactivated by addition of phenol to 0.5% andincubated at room temperature for 2 hours.

[0072] The cells were harvested by centrifugation at 5000×g for 30 minusing 1 liter bottles in a JS 5.2 rotor (Beckman Instruments, Inc., PaloAlto, Calif.). The pelleted cells were combined and weighed. The yieldwas 8 μm packed cells per liter of culture. The packed cells were keptfrozen until further processing.

[0073] B. Preparation of NOMV from Cell Paste

[0074] NOMV was extracted from the cells by a modification of a wellknown method [Zollinger, et al. (1979) J. Clin. Invest. 63:836-848].Briefly, the cells were suspended in 5 volumes of TES buffer pH 7.5 andwarmed at 54-56° C. for 30 min. The cell suspension was then cooled toroom temperature and sheared in a Omnimixer (Dupont Instruments-Sorvall,DuPont Co., Newtown Conn.) at top speed for 3 minutes. The resultingsuspension was centrifuged at 23,500×g for 20 minutes and thesupernatant collected and set aside. The pellets were re-extracted withdistilled water using one-half the volume of the supernatant from thefirst extraction. The cells were not reheated but were sheared in theOmnimixer at top speed for 3 minutes. The cell suspension wascentrifuged at 23,500×g for 20 min and the supernatants combined. Thecell pellets were discarded. The combined supernatants were centrifugedagain at 23,500×g for 15 min and the pellets discarded.

[0075] The extract supernatant was ultracentrifuged at 186,000×g for 60min at 4° C. The supernatant was discarded and the resulting pelletswere thoroughly suspended in distilled water at one-half the originalsupernatant volume. The solution was then centrifuged at 10,000×g for 10minutes to remove any large aggregated material and the supernatantre-centrifuged at 186,000×g for 60 min. The supernatant was discardedand the pellets thoroughly resuspended in one-tenth the original extractsupernatant volume of distilled water. The purified NOMV was filtersterilized and assayed for protein.

EXAMPLE 2

[0076] Intranasal Vaccination of Rabbits With NOMV from Parent (9162)and Sialic Acid Deficient Mutant (9162 synX(−)) Strains of Meningococci.

[0077] The humoral and mucosal response of rabbits to intranasalvaccination with NOMV was investigated using NOMV prepared from strain9162 (Lot D190894) and from the sialic acid deficient mutant of thisstrain 9162 synX(−) (Lot E110195). The second objective was to determineif the presence or absence of capsule and sialylated LOS affected theimmunogenicity of the NOMV.

[0078] Unanesthetized rabbits were intranasally immunized at days 0, 28,and 56. Each of the two NOMV vaccines was administered to four rabbitsat 100 μg protein per dose. An additional group of rabbits receivednormal saline in place of vaccine. Rabbits were weighed to the nearestgram at days 0, 4, and 7 to grossly examine for any acute toxic effectsof the vaccines. Bleeds and nasal washes were taken at days 0, 14, 28,42, 56, and 70. Lung lavages and mouth swabs (to examine salivaryantibody) were done after euthanasia at day 70. Serum bactericidalactivity was assayed using the parent capsule positive strain as thetest strain, and meningococcal-specific serum IgG and IgA was measuredusing an ELISA with lot E110195 NOMV or purified LOS as antigen. Theamount of meningococcal-specific IgA was also measured by ELISA in nasalwash, saliva, and lung lavage samples using lot E110195 NOMV as antigen.

[0079] The mean weight for each grout of rabbits increased at day 4 and7 and no adverse side effects were noted in any rabbits based upon grossobservation. The serum bactericidal antibodies of rabbits immunizedintranasally with the NOMV vaccines increases 30 to 200 fold after threedoses. The bactericidal antibody titers of rabbits vaccinated with lotE110195 NOMV from the synX(−) strain rose sooner and to higher levelsthan rabbits immunized with parent NOMV (FIG. 1). NOMV from bothmeningococcal strains induced a local mucosal antibody response, as seenby the sharply increased levels of anti-NOMV IgA antibodies in nasalwashes (FIG. 2). Specific serum IgG and IgA antibodies increasedstrongly after vaccination with either NOMV vaccine (table 1). Inaddition, increased levels of NOMV specific IgA antibodies were observedin saliva and lung lavage fluids. NOMV from the mutant strain, however,induced the highest amount of lung lavage IgA (table 1). Immunoblottingusing serum from 2 rabbits each ii groups 1 and 2 indicated thatmeningococcal-specific IgG antibodies were directed at a wide variety ofimmunoreactive bands, including class 1, 3, 4, and 5 proteins, LOS, andseveral high molecular weight proteins that may include thetransferrin-binding proteins (data not shown). The antibody response tothe L3,7 and L8 immunotypes of LOS was quantitatively determined byELISA. Although the NOMV vaccine contained predominantly L3,7 LOS, 100to 1000 fold increases in antibodies to both the L3,7 and L8 LOS wereinduced (FIG. 3).

[0080] Conclusions: The NOMV vaccines given intranasally did not causeany detectable weight loss or other acute toxic effects. NOMV from thenoncapsular mutant (synX(−) NOMV) induced a higher bactericidal responsethan NOMV from the encapsulated parent NOMV. Intranasal immunizationswith NOMV induced high levels of meningococcal-specific serum IgG andIgA antibodies by ELISA.

[0081] Intranasal immunization with NOMV vaccines induced a localmeningococcal-specific mucosal response as shown by the high levels ofIgA in nasal wash, saliva, and lung lavage samples. The serum antibodyresponse was directed against a variety of surface antigens, includingthe class 1, 3, 4, and 5 proteins, LOS, and several high molecularweight proteins. TABLE 1 Antibody responses of rabbits to intranasalvaccination with NOMV vaccines by ELISA with synX(-)NOMV used as antigenAntibody level at day 70 after vaccination with indicated vaccineAntibody Parent synx(-) Fluid isotype Saline NOMV NOMV Serum IgG 7.011560 1767 (μg/ml) Serum IgA 6.9 1514 1242 (ELISA units) Lung IgA 0.7671.9 222 washes (ELISA units) Saliva/ IgA 0.39 18 17.7 mouth ELISA swabunits)

EXAMPLE 3

[0082] GMP Production of NOMV, Lot #0123 and Testing for Safety andPotency

[0083] A production lot of NOMV vaccine was produced from strain 9162synX(−) under good manufacturing conditions for testing in humanvolunteers. The vaccine was tested in preclinical safety andimmunogenicity studies.

[0084] Production of Cell Paste. A master seed lot and a production seedlot of N. meningitidis strain 9162 synX(−) were prepared and storedfrozen at −70° C. in Greave's solution. A vial of the production seedwas thawed and used to inoculate 6 agar plates containing GC medium withdefined supplement. The plates were grown overnight at 37° C. in a 5%CO₂ atmosphere and the following day the growth from each two plates wassuspended in 10 ml of sterile, modified Catlin's medium without iron andused to inoculate 1 liter of modified Catlin's medium without iron in a2800 ml Fernbach flask. The three inoculated Fernbach flasks wereincubated on a rotary shaker at 37° C. operating at 180 cycles per minfor 5.5 hours. At the end of this time the optical density of thecultures was determined and the purity of the cultures verfied by Gramstain. The three liters of culture were then used to inoculate 27 litersof modified Catlin's medium without iron in a 40 liter fermenter. Theculture was grown for 3 hours in the 40 liter fermenter and then used toinoculate 270 liters of modified Catlin's medium without iron in a 300liter fermenter. The culture was grown in the 300 liter fermenter for 4hours at which time the culture had reached stationary phase. Theculture was then inactivated by addition of phenol to a finalconcentration of 0.5%. After 2 hours the culture was harvested bycentrifugation in a continuous flow Sharples AS26SP centrifuge(Alfa-Laval Separation, Inc., Warminster, Pa.). The yield was 839 gramsof cell paste which was stored frozen. This was about 30-35% of theyield of a comparable fermentation with excess iron in the medium.

[0085] Preparation of NOMV from Cell Paste. The cell paste (414 gm) wasthawed and suspended in 6 volumes of TES buffer at room temperature. Thesuspension was divided into two 4 liter flasks, placed in a 58° C. waterbath and mixed periodically. After the temperature of the solution hadreached 52 to 54° C. incubation was continued for 30 min. The suspensionwas removed from the water bath, cooled and sheared in 800 ml batches ina 1 liter Waring blender at high speed for 3 min. The suspension wasthen centrifuged at 23,500×g for 20 min using a GSA rotor in a Sorvallrefrigerated centrifuge (Dupont Instruments/Sorvall, Newtown, Conn.).The supernatant was retained and the pellets were resuspended in 2.5 mlof distilled water per gram of cell paste processed. The suspension wassheared in the Waring blender as before and the suspension centrifugedas above at 23,500×g for 20 min. The pellets were discarded and thesupernatants from the two extractions combined. The combined supernatantwas recentrifuged at 23,500×g for 15 min at 4° C. and the pelletsdiscarded. The final combined supernatant was then ultracentrifuged at180,000×g for 60 min to pellet the NOMV. The pellets were resuspended insterile saline equal to one-half the volume of the initial combinedsupernatant before ultracentrifugation. After thoroughly resuspendingthe pellets the solution was centrifuged at 16,000×g for 15 min, thepellets discarded, and the supernatant ultracentrifuged at 180,000×g for60 min. The pelleted NOMV was suspended in a total of 250 ml of sterilesaline. Analysis of the NOMV revealed that residual nucleic acid wasabove 1-2%. Accordingly, the NOMV was diluted to a total of 800 ml withwater for injection and the NOMV pelleted a third time in theultracentrifuge at 180,000×g for 60 min. The pelleted NOMV was suspendedin 450 ml of sterile saline, sterile filtered through a 0.2 μm pore sizemembrane filter and stored at 4° C. The final vialed product wasdesignated Lot #0123 and consisted of 10 ml vials containing 5 ml ofNOMV in normal saline at 800 μg/ml protein, 220 ug/ml LOS. The finalproduct was analyzed by polyacrylamide gel electrophoresis and bynegative stain electron microscopy. The results of these analyses areshown in FIGS. 4 and 5.

[0086] Safety and Toxicity Testing. The final product NOMV lot #0123 wastested for pyrogenicity in rabbits by both the standard intravenous test(Code of Federal Regulations, Title 21, Section 610.13b) and also in amodification of the standard assay where the vaccine was given by theintranasal route (table 2). The NOMV was found to be several thousandfold less toxic when administered intranasally to rabbits than whenadministered intravenously. In the general safety/toxicity test (Code ofFederal Regulations, Title 21, Section 610.11) the NOMV was found to benontoxic at 50 μg in the mouse and 500 μg in the guinea pig. In thelatter test the concentrations tested were based on dosage levels forparenteral vaccines rather than proposed intranasal dosages. TABLE 2Results of safety testing of Lot #0123 of NOMV vaccine. Test Dose ofNOMV Result Standard Rabbit 0.1:μg per Failed Pyrogen Test rabbit(Intravenous) Standard Rabbit 0.05:μg per Passed Pyrogen Test rabbit(Intravenous) Intranasal 100:μg per Passed Rabbit Pyrogen rabbit TestIntranasal 100:μg per Passed Rabbit Pyrogen rabbit Test (24 hours)Non-GLP 400:μg per Passed Intranasal rabbit Rabbit Pyrogen General 50 μgPassed Safety/Toxicity in Mice General 500 μg Passed Safety/Toxicity inGuinea Pigs

[0087] Potency Testing of NOMV Lot #0123 in Mice. Fifteen groups of 5outbred CD1 white mice were vaccinated with NOMV vaccine Lot #0123.Groups 1 to 7 were vaccinated intranasally with 25 μl of vaccinecontaining dosages of 0.03, 0.10, 0.30, 1.0, 3.0, 10, or 20 μg ofvaccine (protein). Five mice were given each dosage. Groups 9 to 15 werevaccinated intraperitoneally with 0.1 ml of vaccine containing 0.01,0.03, 0.1, 0.3, 1.0, 3.0, or 10 μg of vaccine (protein). Group 8received normal saline in place of vaccine. The indicated dosages weregiven at 0 and 28 days, and blood was drawn on days 28 and 42. Thelevels of antibodies in the sera were determined and compared to thegroup that received saline in place of vaccine. The sera were assayed bybactericidal assay which is a functional assay that has been correlatedwith protection and also by ELISA for total IgG and IgA antibodies tohomologous NOMV. Both routes of immunization induced high titers ofbactericidal antibodies to the encapsulated parent strain 9162, butintranasal vaccination required about 10-fold more antigen to achieve acomparable antibody response (FIGS. 6 and 7). ELISA assays also revealedthat intranasal vaccination induced high levels of specific IgGantibodies (FIGS. 8 and 9) and IgA antibodies (FIGS. 10 and 11). Higherlevels of IgA antibodies were induced with intranasal vaccination ascompared to intraperitoneal vaccination, but lower levels of IgGantibodies were induced.

EXAMPLE 4

[0088] Improved Method for Large Scale Preparation of NOMV

[0089] Shake flask cultures of N. meningitidis strain 9162 synX(−) weregrown and harvested as described in example 1. The procedure asdescribed in example 1 was followed up to the first ultracentrifugationstep. At this point in the process the crude extract was split in twoparts. One part of the extract was processed as described below usingsteps that were substituted for the two ultracentrifugation steps(180,000×g for 60 min at 4° C.) used in examples 1 and 2 as a means ofpurifying the NOMV from the cell extract. The other part of the extractwas processed by ultracentrifugation as described in example 1.

[0090] An amount of Whatman DE52 DEAE cellulose equal to two grams per10 ml of cell extract was washed twice in about 10 volumes of TESextraction buffer and collected on a filter paper using a Buchner funnelfitted in a vacuum side arm flask. The cake of washed DEAE cellulose wasthen added to the cell extract and mixed at room temperature for 1 hour.After this time the DEAE cellulose was removed by passing the suspensionthrough a Buchner funnel fitted with a suitable filter paper. Thefiltered solution was recovered and kept for further processing. Afterthis step the extract was found to be essentially free of nucleic acid.Other DEAE ion exchange matricies may also be used in this procedure.

[0091] The extract was then processed by ultrafiltration using a 500,000MWCO membrane or by microfiltration using a 0.1 μm pore size membrane(A/% Technologies Corp. Needham, Mass.) cartridges UFP-500-C4A andCFP-1-D-4A respectively. The ultrafiltration (or microfiltration) columnwas set up, cleaned, and used according to the manufacturer'sinstructions. The extract was added to the ultrafiltration system andcycled through the column using about 2 to 15 PSI back pressure andkeeping the volume constant by continuous addition of 0.9% sodiumchloride solution. The process was continued until 20 volumes of sodiumchloride solution had passed through the membrane. The retentate wasthen concentrated to one tenth the original volume. The process wasstopped and the retentate recovered from the column. Table 3 and FIG. 12show the comparative results obtained with the DEAE/ultrafiltrationmethod as compared to the normal ultracentrifugation method. Theultrafiltration cartridge and the microfiltration column gave similarresults, although the flow rate was better with the ultrafiltrationcolumn. The data in table 3 and FIG. 12 show that the yield, compositionand purity were very similar indicating that the new method could beused as an adequate substitution for the normal ultracentrifugationmethod. TABLE 3 Comparative results of two methods for purifying theNOMV from crude cell extract. Ultracen- trifuga- DEAE/Ultrafil- tiontration Crude 2 3 0.1 μm Measure- ex- cy- cy- 500,000 pore ment tractcles cles MWCO size Protein 380 44.3 40.2 46.6 47.0 concentra- tionNucleic 144 1.66 0.68 0.8 0.8 acid concentra- tion OD_(280 nm)/ 0.540.96 1.23 1.21 1.21 OD_(260 nm)

EXAMPLE 5

[0092] Preparation of NOMV from E. coli lpp(−), ompA(−) Double Mutant

[0093] The feasibility of extending the NOMV vaccine approach to othergram negative organisms that, unlike Neisseria spp., do not have a looseouter membrane that naturally blebs off during growth was demonstratedby applying the method of preparing NOMV from the meningococcus to adouble mutant of E. coli that is lacks both the Braun lipoprotein (Lpp)and OmpA.

[0094] The strain was streaked for isolation and grown at 37° C.overnight on Luria agar plates containing 30 mM MgCl₂. The growth fromthe plate was transferred to a one liter flask containing 200 ml ofliquid Luria broth containing 30 mM MgCl₂ and grown overnight on arotary shaker at 37° C. The purity and spherical morphology of the cellswere verified by Gram stain. The cells were harvested by centrifugationat 16,000×g for 15 min, and NOMV harvested essentially as described forN. meningitidis in example 1 but modified for smaller scale. The cells(1.5 grams) were suspended in 20 ml of TES buffer at pH 7.5 and heatedat 60° C. for 30 min. The suspension was then sheared in the Omnimixerat 90% of maximum speed for 1 min and the cells spun out bycentrifugation at 16,000×g for 15 min. The supernatant was recovered andset aside, and the pellets were resuspended in 10 ml of TES buffer andreextracted by the same procedure. The two supernatants were combined.The NOMV were isolated from the crude extract by ultracentrifugation at186,000×g for 75 min. The supernatant was discarded and the pelletsuspended in 10 ml of distilled water and centrifuged at 16,000×g for 15min. The low speed pellet was discarded and the supernatant was thenultra centrifuged at 186,000×g for 75 min. The resulting NOMV pellet wasrecovered and suspended in 2 ml of distilled water and filtered througha 0.45 μm pore size membrane filter.

[0095] The NOMV was assayed for protein and the yield was calculated tobe 3 mg of NOMV protein per 200 ml culture or 2 mg NOMV protein per gramof packed cells. This is approximately the same yield that is obtainedfrom N. meningitidis cells. The E. coli NOMV was analyzed bypolyacrylamide gel electrophoresis, and the results are given in FIG.13. The protein band pattern is quite simple as expected for an outermembrane preparation with two deleted proteins. One major band is seen,probably a porin, and a number of minor bands. Further analysis of theE. coli NOMV is required, but these results provide preliminary evidencefor the feasibility of extending the NOMV approach to other Gramnegative bacteria.

What is claimed is:
 1. An immunogenic composition for the immunizationof an individual comprising native outer membrane vesicles of Gramnegative bacteria.
 2. An immunogenic composition according to claim 1wherein, said Gram negative bacteria is Neisseria.
 3. An immunogeniccomposition according to claim 2 wherein, said Neisseria is N.meningitidis.
 4. An immunogenic composition according to claim 3wherein, said N. meningitidis is N. meningitidis Group B.
 5. A methodfor preparing native outer membranes from Neisseria, said methodcomprising the steps of: (i) shearing Neisseria cells; (ii) separatingcells and cellular debris from native outer membrane vesicles; (iii)passing said native outer membrane vesicles through an ion exchangematrix; and (iv) ultrafiltering said native outer membrane vesicles. 6.A strain of N. meningitidis Group B which is unable to synthesize sialicacid.
 7. A strain of N. meningitidis according to claim 6 wherein, saidstrain expresses iron uptake proteins.
 8. A strain of N. meningitidisaccording to claim 7 wherein, said strain is 9162 synX(−).
 9. A methodfor producing a strain of N. meningitidis which expresses iron uptakeproteins said method comprising growing said N. meningitidis in irondeficient media.
 10. A vaccine for protection against infection withGram negative bacteria, said vaccine comprising native outer membranevesicles of said bacteria in an amount effective to elicit protectiveantibodies in an animal to said Gram negative bacteria, and apharmaceutically acceptable diluent.
 11. A vaccine for protectionagainst infection with Gram negative bacteria according to claim 10,wherein said bacteria is Neisseria.
 12. A vaccine for protection againstinfection with Gram negative bacteria according to claim 11 wherein,said Neisseria is N. meningitidis.
 13. A vaccine for protection againstinfection with Gram negative bacteria according to claim 12 wherein,said N. meningitidis is N. meningitidis Group B.
 14. A vaccine forprotection against Gram negative bacteria according to claim 13, whereinadministration of said vaccine is intranasal.
 15. A biologically purenative outer membrane vesicles composition comprising native outermembrane vesicles from a strain of Neisseria according to claim
 6. 16. Abiologically pure native outer membrane vesicles composition accordingto claim 15, wherein said Neisseria strain further expresses iron uptakeproteins.
 17. A treatment for reducing N. meningitidis infectionsymptoms, said method comprising administering to a patient in need ofsuch treatment an effective amount of antibodies against native outermembrane vesicles of N. meningitidis in a pharmaceutically acceptableexcipient.